High sensitivity d1000 screentape and reagents

100 ng of DNA was aliquoted from each sample. Unmethylated λDNA was added to each sample at 0.5%w/v and the samples were sheared mechanically using a Covaris LE220-plus system to a length of 350 bp, using the settings recommended by the manufacturer. The sizing was determined by a High Sensitivity D1000 ScreenTape and Reagents (Agilent, Cat. No. 5067-5584 and 5067-5585) on the TapeStation platform. Once the input DNA was at the proper fragment size, the samples were concentrated with a SpeedVac to a volume of 20µL. The samples then underwent bisulfite conversion with an EZ DNA Methylation- Gold kit (Zymo, Cat. No. D5006). The samples were eluted off the spin columns with 15 μl of low EDTA TE buffer (Swift, Cat. No. 30024) before library preparation.
The low-input libraries were prepared using an ACCEL-NGS Methyl-Seq Library kit (Swift, Cat. No. 30024) with a Methl-Seq Set A Indexing Kit (Swift, Cat. No. 36024), following the protocol associated with the library kit. During the protocol, bead cleanup steps were performed with SPRIselect beads (Beckman Coulter, Cat. No. B23318). Following the recommendation of the kit, 6 PCR cycles were performed to amplify the samples. The final libraries were quantified with a Qubit dsDNA HS Assay Kit and the size was determined by using a BioAnalyzer High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626).

After PCR amplification, the dsDNA HS assay kit for the Qubit 3.0 fluorometer (Thermo) and the TapeStation 4200 with the High Sensitivity D1000 ScreenTape and Reagents (Agilent) were used to measure DNA concentration and amplicon lengths, respectively. Tagmentation, clean‐up, normalization and pool of libraries were performed following the Nextera XT DNA Sample Preparation Reference Guide (Illumina) to generate paired‐end DNA libraries. Purification steps were carried out by using AMPure XP beads (Beckman Coulter), and all the resulting indexed libraries were checked on TapeStation. A sequencing run of 2 × 151 bp paired‐end reads was performed on the NextSeq 550 instrument (Illumina). After the procedure, the software generated analysis output in the FASTQ file format.

Input and immunoprecipitation samples were prepared for sequencing with Rnase-free reagents, under Rnase-free conditions, and on ice, using the NEXTFlex Small RNA Sequencing Kit v3 (Perkin Elmer; D-mark Biosciences) according to the manufacturer’s protocol, with the recommended “No Size Selection Protocol” modification for bead cleanup, and using PAGE Size-selection and Cleanup to extract library DNA between 150-170nt in length. Sequencing libraries were quantified using a Qubit 4.0 fluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and Qubit 1x dsDNA HS assay (Invitrogen). The library fragment length distributions were determined using a TapeStation 4150 instrument (Agilent, Santa Clara, CA, USA) with High Sensitivity D1000 ScreenTape and reagents (Agilent). The barcoded libraries were pooled equimolar and 76 bp or 38 bp paired-end reads generated on a NextSeq 550 instrument (Illumina, San Diego, CA, USA).